mRNA decapping by ApaH like phosphatases

• Applicant: Professorin Dr. Susanne Kramer
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Parasitology and Biology of Tropical Infectious Disease Pathogens ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 398051313

• Final Report Year: 2024

Final Report Abstract

The 5´end of a eukaryotic mRNA is usually protected by a m7G cap, a methylated guanosine that is connected to the first transcribed nucleotide via a triphosphate bridge. Special enzymes have evolved to remove this cap during mRNA degradation. In the 5´-3´decay pathway, the canonical decapping enzyme is the nudix hydrolase DCP2; the enzyme works in a complex with several other cofactors. Kinetoplastida separated very early from the major group of eukaryotes and lack homologous to all components of the canonical DCP2 decapping complex. Instead, we had found that they use the ApaH-like phosphatase ALPH1, a bacterialderived pyrophosphatase of the protein phosphatase group that is not related to nudix hydrolases. ALPH1 consists of a catalytic domain and Kinetoplastida-unique N- and C-terminal extensions. In this project, we have done a detailed characterization of ALPH1. We have established a range of in vitro decapping assays and characterized substrate specificity and preferred reaction conditions for ALPH1 in great detail. We found that enzyme activity is solely dependent on the catalytic domain and that ALPH1 has a broad substrate range including cap analogues with a clear preference to unmethylated caps. This unusual substrate preference may reflect the evolutionary origin of ALPH1 from the bacterial ApaH. We have further defined the ALPH1 interaction partners using a combination of BioID and immunoprecipitations with different ALPH1 truncations as baits. We identified the core-decapping complex, consisting of five proteins, including ALPH1 and the downstream 5´-3´ exoribonuclease XRNA, that all share an unusual localization to the posterior pole of the cell. We have done a screen for ApaH like phosphatases across eukaryotes and found that mRNA decapping by this enzyme group is likely unique to Kinetoplastida, as other eukaryotes either of no ALPH proteins, or only very short ones with non-cytoplasmic localization predictions. Our data are the base for a target-based drug screen that aims to treat the neglected tropical diseases caused by Kinetoplastida (African sleeping sickness, Leishmaniasis, Chagas disease) and for a better understanding of the regulation of mRNA degradation in Kinetoplastida. Moreover, ApaH like phosphatases can be considered as “neglected enzymes”: they are widespread among eukaryotes (albeit absent from mammals), but with the exception of Ppn2 from yeast none have been functionally characterized. We are working on closing this gap.

Publications

• The complex enzymology of mRNA decapping: Enzymes of four classes cleave pyrophosphate bonds. WIREs RNA, 10(1).
  Kramer, Susanne & McLennan, Alexander G.
  
• Is mRNA decapping by ApaH like phosphatases present in eukaryotes beyond the Kinetoplastida?. BMC Ecology and Evolution, 21(1).
  Castañeda, Londoño Paula Andrea; Banholzer, Nicole; Bannermann, Bridget & Kramer, Susanne
  
• A unique mRNA decapping complex in trypanosomes. Nucleic Acids Research, 51(14), 7520-7540.
  Kramer, Susanne; Karolak, Natalia Katarzyna; Odenwald, Johanna; Gabiatti, Bernardo; Castañeda, Londoño Paula Andrea; Zavřelová, Anna; Freire, Eden Ribeiro; Almeida, Kayo Schemiko; Braune, Silke; Moreira, Claudia; Eder, Amelie; Goos, Carina; Field, Mark; Carrington, Mark; Holetz, Fabiola; Górna, Maria Wiktoria & Zoltner, Martin